We propose to develop a tetravalent dengue (DEN) vaccine using a novel strategy that will ensure broad protection against DEN virus infection. The disease dengue is caused by four mosquito-borne serologically related flaviviruses and development of a DEN vaccine has been particularly challenging because sequential infection by different DEN serotypes/viruses can lead to DEN hemorrhagic fever (DHF) or dengue shock syndrome (DSS), both of which are life-threatening. As a consequence of this phenomenon, an efficacious and safe vaccine needs to be tetravalent and provide protective immunity simultaneously against all four DEN viruses. Attempts at developing a tetravalent live or attenuated vaccine have thus far failed due to problems with interference, poor immunogenicity and generation of an imbalanced immune response. Here, we present an innovative DEN vaccine strategy designed to overcome these limitations. Our approach involves targeting a mixture of recombinant proteins to antigen-presenting cells by linking a protective antigen to a Toll-like receptor (TLR) ligand. Engagement of a TLR with its associated ligand triggers the innate immune system and initiates a series of events that impact the adaptive immune response. This eliminates the need for adjuvants or carriers typically used in vaccine formulations and increases vaccine potency by enhancing immunogenicity without increasing toxicity. With this strategy, we have successfully developed influenza and West Nile (WN) vaccine candidates that can be produced economically, induce potent antibody responses, are efficacious in mouse models and do not require adjuvant. The goal of this proposal is to develop a tetravalent DEN vaccine by engineering four monovalent fusion proteins comprised of the DEN EIII domain of the envelope protein from each DEN virus fused to bacterial flagellin, a potent TLR5 ligand. To assess induction of DEN virus type-specific neutralizing antibodies, the efficacy and specificity of the immune response to each monovalent vaccine candidate will be examined in mice. These monovalent studies will guide identification of a preclinical tetravalent formulation that will involve a mixture of all four fusion proteins at ratios necessary to induce an equitable immune response. A tetravalent combination will be optimized in mice and tested in a non-human primate challenge model as proof-of-concept. We hypothesize that a cocktail formulation of all four monovalent proteins will induce a balanced tetravalent immune response and provide type-specific protection against all four DEN viruses without the risk of producing antibodies that may enhance DEN infection and lead to DHF. [unreadable] [unreadable] [unreadable]